Overall Summary ? Project 3 There is resurgent interest in immunotherapy approaches against glioblastoma multiforme (GBM), a brain cancer with dismal prognosis. We have pioneered the utilization of Gene-Mediated Cytotoxic Immunotherapy (GMCI), consisting of tumor injection of aglatimagene besadenovec (AdV-tk) a non-replicating adenovirus expressing the Herpes virus thymidine kinase gene followed by an oral anti-herpetic prodrug (valacyclovir), combined with standard of care (SOC) in humans with newly diagnosed GBM. Mature phase II data appears to show encouraging, albeit not definite, results in terms of possible efficacy based on the extent of residual tumor burden. Our pilot data shows that efficacy may be limited by immune checkpoint signaling networks, such as PD-1/ PD-L1. Therefore, our overarching hypothesis is that a combination of GMCI-based intratumoral immunostimulation with inhibition of immune checkpoint signaling will provide increased effectiveness in animal models of glioma and in a planned prospective randomized clinical trial. In addition, we hypothesize that extracellular vesicles (EVs) and their contents released by glioma cells provide immunomodulatory signals in the GBM microenvironment. These will be further studied in an integrated mode with the other Project 1 and 2 of this PO1. Core B and C will be utilized as well for animal models and sample procurement/storage. These hypotheses will be tested by accomplishing the following specific aims: Aim 1: Characterize the impact of GBM tumor burden on the efficacy of GMCI alone and in combination with immune checkpoint inhibition. Hypothesis: GBM tumor burden mitigates the impact of GMCI due to cytotoxic T cell exhaustion. Significance: Our preclinical and clinical data suggests that GMCI is most effective with minimal tumor. However, the impact of Immune checkpoint inhibition on this effectiveness is not known. Impact: This knowledge would help in the design of our planned clinical trial for GBM. Aim 2: Determine if GMCI can sensitize resistant GBMs to immune checkpoint inhibition. Hypothesis: The response of different GBMs to GMCI depends on their mutational load and/or differing expression of immune checkpoint activating ligands. Significance: Since our preclinical data shows that not GBM models differ in response to PD-1 immune checkpoint inhibition, these experiments would determine what other immune checkpoint signals may be operative and/or whether altering the mutation load in different GBM cells may lead to different efficacy responses. Impact: This knowledge would also help in clinical trial design and provide mechanistic evidence of possible resistance mechanisms. Aim 3: Determine the significance of immune checkpoint inhibitory protein expression in extracellular vesicles (EVs) released by GSCs after GMCI in vitro and in vivo. Hypothesis: Immune checkpoint inhibitory proteins in EVs are a novel mechanism of glioma immuno-evasion after therapy and may help identify patients responsive to this therapy. Significance: Expression of PD-L1 and other immune checkpoint inhibitory proteins in EVs will help tumor immuno-evasion, and provide potential biomarkers for therapy. Impact: This knowledge would impact future design of immunotherapy trials.